Since the pioneering studies of McGee and co-workers using fallopian tube explants, the concept that Neisseria gonorrhoeae can invade nonprofessional phagocytic cells as part of its pathogenesis has gained acceptance. Using immunoelectron and confocal microscopy, we have recently shown in urethral exudates from infected males that N. gonorrhoeae invade urogenital cells lining the urethra. Gonococci in these cells are found primarily n vesicles and occasionally free in the cytoplasm. Acridine orange staining of coiled DNA indicates the gonococci are alive within these urogenital cells. Serial sections suggest that organisms can be released from these vesicles. In this proposal, we wish to address the hypothesis that an intracellular existence in the urethral stratified squamous epithelial cell is an integral factor in disease pathogenesis. To accomplish this goal, we will determine the mechanisms by which gonococci gain access to the intracellular environment of these cells and what factors determine their compartmentalization within the cell. This will be achieved through the following specific aims: 1) To study the biology of attachment and invasion of primary cell cultures of urethral stratified squamous epithelial cells by Neisseria gonorrhoeae strain 1291. These studies will be performed using confocal, electron microscopic and Northern blot analysis and examine such factors as actin polymerization, microtubule formation, Ca2+ flux, vacuole acidification, and asialoglycoprotein expression. Experiments will also be done using a series of genetically and structurally defined LOS mutants of strain 1291 to determine the role of gonococcal LOS in the adherence to and invasion of these cells. 2) To identify the genes in Neisseria gonorrhoeae which are responsible for inhibition of vacuole acidification in stratified squamous epithelial cells. This will be accomplished with: A) a genomic library of mTn3 and/or Tn10 mutants of Neisseria gonorrhoeae strain 1291 which will be used to infect primary stratified squamous epithelial cell cultures. Flow cytometric separation of these infected urethral stratified squamous cells will be used to segregate cells containing organisms which inhibit vacuole acidification. B) the development of a pIVET system for use in N. gonorrhoeae which will determine if the gene(s) associated with inhibition of vacuole acidification are regulated intracellularly.